Streptokinase (SK) is a secretory protein of hemolytic Streptococcus. It is a thrombolytic agent currently used to treat vascular thromboembolic symptoms such as acute myocardial infarction. SK can activate human plasminogen (HPlg) to human plasmin(HPlm), which is a serine protease. The HPlm then directly catalyzes the hydrolysis of fibrin in the blood clots to relief or to prevent thromboembolic diseases.
However, SK is a labile protein susceptible to degradation in reaction with HPlm. The HPlm-degraded SK fragments had lower activities as a HPlg activator in comparison with the native SK Shi et al. (1994), Biochem. J. 304: 235-241!. It is therefore extremely desirable to construct SK mutants which are capable of activating HPlg to HPlm, yet are resistant to Hplm degradation.
The peptide bonds of the SK molecule that were hydrolyzed by HPlm were previously determined See Shi et al. (1994), cited above!. The plasmin specifically catalyzes the hydrolysis of peptide bonds having at the amino side Lys and Arg. More specifically, the peptide bond Lys59-Ser60 of SK is among the few peptide bonds which are cleaved in the early reaction with HPlm while the NH2-terminal peptide, Ile1-Lys59, is essential in stabilizing the structure of SK See Shi et al. (1994) cited above!. Therefore, a more stable SK mutant can be constructed by site-directed mutagenesis or other amenable genetic cloning techniques in that the early hydrolysis of the peptide bond Lys59-Ser60 by HPlm can be prevented.
Additionally, the SK mutants which are more stable in reaction with Hplm may potentially be better thrombolytic agents than the native SK in treatment of thromboembolic disease. The improved mutants can also be used to form a HPlg+SK complex, such as acylated derivatives of HPlgSK complex, as a slow activated thrombolytic agent Smith et al. (1981), Nature, 290: 505-508!. The total or partial sequences of the modified SK can also be incorporated in a complex molecule, such as SK and anti-fibrin specific antibody (59D8) fusion protein Hui et al.(1983), Science, 222: 1129-1132! for therapeutic purposes.
It is therefore an objective of the invention to demonstrates a novel way to create mutants of SK that are more stable in reaction with HPlm and have better thrombolytic efficacy.
It is a further objective of the invention to construct SK mutants resistant to HPlm hydrolysis by gene cloning techniques to selectively modify one or more amino acids of SK at or near the cleaved sites.